The major purpose of these studies is to determine the molecular mechanisms by which insulin-like growth factor binding protein-5 (IGFBP- 5) functions to modulate IGF actions on fibroblasts and to determine if any of its functions are altered in fibroblasts derived from older age donors. IGFBP-5 has been shown to modulate IGF bioactivity in fibroblasts. IGFBP-5 is synthesized and secreted by fibroblasts, and its capacity to modulate IGF actions is regulated by specific, post- translational modifications. The major modifications appear to be proteolysis, which destroys IGF-I binding, phosphorylation which enhances its affinity for IGF-I, and adherence to extracellular matrix, which results in the diminution in affinity, but a potentiation of cellular responsiveness to IGF-I. These studies will focus on defining mechanisms that control these posttranslational modifications and how they function to alter IGF action on this cell type. The site of proteolysis will be determined using carboxyterminal sequencing and in vitro mutagenesis. Mutants with an altered proteolytic cleavage site will be used to study the functional consequences of creating protease-resistant forms. The factors that control the release of proteolytic activity from fibroblasts will be analyzed and the mechanism by which IGF-I and II binding to IGFBP-5 results in resistance to proteolysis will be defined. Further studies will define the role of adherence of IGFBP-5 to extracellular matrix (ECM) in controlling IGF bioactivity in fibroblasts. The ECM binding site within the IGFBP-5 molecule will be ascertained by carboxyterminal sequencing of peptides that have variable degrees of adherence to ECM. The sites that are necessary for ECM binding activity will be altered by mutagenesis and the mutagenic CDNA'S that result in the greatest loss of ECM binding will be transected into human fibroblasts. Clones will be isolated that secrete high concentrations of the wild type and mutant forms, and the cellular replication response to IGF-I will be determined using both cell types. If loss of IGFBP-5 binding to ECM is associated with a diminution in the response to IGF-I, the functional consequences of this reduction on IGF-I receptor function will be determined. Receptor down regulation and signal transduction as mediated through PP 175 phosphorylation and c-ras induction will be studied as markers of receptor function. The role of phosphorylation of IGFBP-5 in controlling its bioactivity will be analysis. The positions of serines and threonines in IGFBP-5 that are phosphorylated will be determined and mutants with these sites of phosphorylation altered will be used to determine if loss of phosphorylation alters the affinity of IGFBP-5 for IGF-I. The role of these mutations in altering IGFBP-5 bioactivity will be determined by assessing the effects of the pure mutagenized protein on IGF-I responsiveness. The kinase that is responsible for IGFBP-5 phosphorylation in fibroblasts will be characterized and the factors that regulate kinase activity determined. Since dephosphorylation of IGFBP-5 may result in potentiation of IGF action, the presence of phosphatase activity will be determined and if present whether it is altered by growth factors that alter fibroblast responsiveness to IGF-I. The results of these studies should define the molecular mechanisms by which IGF-I functions with IGFBP-5 to stimulate fibroblast replication and may suggest novel strategies for determining the mechanisms by which fibroblasts from older age donors lose their responsiveness to this growth factor.